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Highly sensitive LC-MS/MS workflow for targeted

quantification of host cell proteins

Featuring the SCIEX QTRAP® 6500+ LC-MS/MS System

Lei Xiong1, Ian Moore2 and Ji Jiang1 1SCIEX, Redwood City, CA, USA 2SCIEX, Concord, ON, Canada

During biotherapeutics manufacturing, process-related impurities

and other trace contaminants accompany the recombinant

biotherapeutic products. Among them, host cell proteins (HCPs)

are a major class of protein impurity derived from the host

organism. The detection and quantification of HCPs is an area of

particular concern, as these contaminants can elicit an adverse

response in patients.

HCP quantification methods have been adopted in

manufacturing and quality control processes in

biopharmaceutical industry in recent years. The high complexity

and the wide dynamic range of protein concentrations in the

multiple purification stages of biotherapeutic production pose

challenges for the traditional data dependent workflows for HCP

quantification. This results in the increasing need for developing

targeted HCP analysis methods with high sensitivity, reduced

analysis time, robustness and multiplexing capability (quantify

significant numbers of analytes in one injection). As the target

analytes for quantification have been pre-defined before

analysis, triple quadrupole and QTRAP LC-MS/MS Systems are

identified as the suitable instrument platforms do to their high

quantitative performance. Herein, a targeted HCP analysis

workflow utilizing the SCIEX QTRAP 6500+ LC-MS/MS System

is presented. A Scheduled MRMTM Algorithm is applied to allow

the simultaneous quantification of 48 proteins (4 transitions per

protein) in an 8 min LC-MS analysis. This method demonstrated

LLOQs ranging from 0.09 to 5 ppm.

Key features of the LC-MRM based targeted HCP quantification workflow

• The SCIEX Triple Quad™ and QTRAP 6500+ Systems offer

superior quant performance for bioanalysis, reaching low

LLOQs (sub ppm level HCP quantification), wide LDR and low

CVs

• High analysis throughput to quantify 48 proteins in an 8 min

LC-MS/MS run with solid confirmation (4 transitions per

protein)

• High-resolution MS/MS peptide library offering reliable

signature peptide selection using the SCIEX QTRAP 6500+

LC-MS/MS System

• Seamless integration of Skyline with SCIEX Software allowing

fast and robust MRM method optimization

• Comprehensive automated quantification with SCIEX OS-MQ

Software combining ease-of-use with confidence in the results

Figure 1. The extracted ion chromatograms (XICs) of 96 peptides (two MRM transitions per protein) from 48 target proteins demonstrate the LC-MRM based targeted HCP quantification workflow. The total LC run time is 8 min, with a 5 min linear gradient for separation.

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Methods

Sample preparation: NISTmAb monoclonal antibody

(NISTmAb) and the Universal Proteomics Standard (UPS) are

purchased from Sigma-Aldrich. In this experiment model,

NISTmAb is serving as the biotherapeutic molecule, while the 48

human proteins in the UPS mix are mimicking the targeted HCPs

for quantification. Bovine serum albumin (BSA) is used as the

internal standard. The UPS proteins and BSA are spiked into

NISTmAb solution and serial diluted. In the serial dilution

samples, the level of BSA remains consistent as 100 ppm, and

the level of UPS proteins ranges from 1.22 to 5000 fmol per 100

µg NISTmAb. The ppm level range for each individual protein

varies based on the protein molecular weight (MW). For the

smallest protein, epidermal growth factor (P01133) with MW

6353 Da, its concentrations are 0.08 – 317.65 ppm among serial

dilution. For the largest protein, gelsolin (P06396) with MW

82959 Da, its concentrations are 1.01 – 4147.95 ppm.

Samples are denatured by incubating with N-octyl-glucoside

(OGS), reduced by dithiothreitol (DTT) and alkylated by

iodoacetamide (IAM). A trypsin/Lys-C digestion was performed

at 37 °C for overnight, with an enzyme-protein ratio at 1:25.

Formic acid was spiked into the samples to abort digestion. The

samples are centrifuged at the speed of 12000 g and injected

into LC-MS analysis.

LC-MS conditions: Two types of LC-MS analysis are performed

either on a TripleTOF® 6600+ LC-MS/MS System to build

peptide library, or on a SCIEX QTRAP 6500+ LC-MS/MS

System for protein quantification.

Peptide library creation: An information dependent acquisition

(IDA) analysis is performed to analyze the digested NISTmAb-

UPS protein mix. The LC-MS details are summarized in Table 1

and 2.

Protein quantification: A Scheduled MRM Algorithm analysis is

performed on the serial dilution samples for protein

quantification. The LC-MS details are summarized in Table 3,

Table 4 and Table A1.

Data processing: Figure 2 describes the workflow for data

generation and processing.

Peptide library creation: The database search is performed on

IDA data by using ProteinPilot™ Software 5.0. A .fasta file

including sequences of NISTmAb, BSA and USP proteins is

Table 1. HPLC condition for IDA analysis.

Parameter Value

Stationary phase C18 column, 2.1 X 150 mm, 1.7 µm

Mobile phase A 0.1% formic acid in water

Mobile phase B 0.1% formic acid in acetonitrile

Gradient B% ramping from 5% to 40% in 42 min

Total run time 60 min including equilibration

Flow rate 0.2 mL/min

Column temperature 40 °C

Injection volume 20 µL

Table 2. Mass spectrometric parameters for IDA analysis.

Parameter Value Parameter Value

MS range 350-1500 MS/MS range 150-1500

MS accumulation time

150 ms MS/MS accumulation time

30 ms

Curtain gas: 30 psi Source temperature: 450 °C

Ion source gas 1: 50 psi Ion source gas 2: 50 psi

Number of MS/MS triggered per circle

25 Ion spray voltage: 5500 V

Table 3. HPLC condition for MRM analysis.

Parameter Value

Stationary phase Phenomenex Kinetex C18 column, 3 X 50 mm, 2.6 µm

Mobile phase A 0.1% formic acid in water

Mobile phase B 0.1% formic acid in acetonitrile

Gradient B% ramping from 12% to 32% in 5 min

Total run time 8 min including equilibration

Flow rate 0.5 mL/min

Column temperature 40 °C

Injection volume 20 µL

Divert valve set-up 1-6.2 min to MS

Table 4. Gas/source parameters for MRM analysis.

Parameter Value Parameter Value

Curtain gas: 30 psi Source temperature: 550 °C

Ion source gas 1: 65 psi Ion source gas 2: 65 psi

CAD gas: 12 psi Ion spray voltage: 5500 V

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created for database search. The search result file is generated

and imported into Skyline software for peptide library creation.

MRM method development: Skyline software is used for MRM

method optimization. The UPS protein sequences are imported,

and the MRM transitions are predicted based on peptide and

transition settings. The most abundant fragment ions observed in

the MS/MS spectra of the peptide library are selected for further

MRM optimization, including retention time verification, and the

optimization of CE, DP, gas/source parameters.

Protein quantification: The optimized MRM method is used for

creation of the calibration curve. The MRM data are processed

by using the Analytics function in SCIEX OS Software 1.7.

Figure 3. Example database search result shown in ProteinPilot Software 5.0. The result file provides information including (from top to bottom panel): the list of identified protein, the summary of peptides identified per protein, the sequence coverage map, the MS/MS fragment ion assignment for each peptide.

Figure 2. The data generation and processing workflow of targeted HCP quantification. Four major steps are involved: peptide library build-up, MRM method development, data acquisition and quantitative data processing. Details and zoomed-in figure are provided in later sessions. Among them, peptide library creation step can be simplified as in-silico digestion, if a high-resolution MS (HRMS) is not available to generate IDA data.

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Peptide library creation

For any signature peptide-based protein quantification workflow,

selecting the appropriate peptide targets is a critical requirement

to ensure the assay’s success. Several criteria for signature

peptide selection are commonly applied: 1) the uniqueness of

peptide sequences: selected peptides should be unique from any

matrix protein to minimize endogenous interference; 2) the

abundance of LC-MS/MS signal: selected peptides should have

good ionization and fragmentation efficiency for the optimal

assay sensitivity; 3) no missed cleavages or variable post-

translation modification (PTM) site: the peptides should include

neither missed cleavages, nor amino acids (e.g. methionine) that

can be easily modified during the biological process or sample

preparation.

There are two methods commonly used for signature peptide

selection: in-silico digestion of the target protein and IDA

analysis for peptide mapping on the authentic protein digest

sample. When an HRMS instrument is available for IDA analysis,

the later method is usually adopted for the most appropriate and

reliable peptide selection based on real data. Herein, an IDA

peptide mapping analysis is performed on a TripleTOF 6600+

LC-MS/MS System to analyze the digested NISTmAb-UPS

protein mix. The IDA data is processed by ProteinPilot Software

5.0 (Figure 3), searched against the protein sequences of

NISTmAb, UPS proteins and BSA. All 48 UPS proteins, together

with BSA and NISTmAb, are identified during database search.

The information of peptide ID, sequence coverage and MS/MS

fragment ion assignment can be found in the search result file.

This result file is imported into Skyline software, served as the

peptide library.

MRM method development

The combination of Skyline software (Version 20.1.0.31) (Figure

4) together with Analyst® Software 1.7 is adopted for MRM

development. After protein sequences are imported into Skyline,

the list of peptides is generated based on peptide settings and

peptide library matching. The MRM transitions are created by

selecting the fragment ions with the strongest MS/MS signal per

peptide in the peptide library. The MRM acquisition method can

be generated in Skyline and imported into Analyst Software 1.7.

The method format can be standard MRM, Scheduled MRM

Algorithm Pro with expected retention time for each peptide

defined, or MRM for compound parameter optimization. The data

generated by using these methods can also be imported back to

Skyline for MRM method optimization, by identifying the optimal

CE and DP values, or adjusting the expected analyte retention

time.

Figure 4. A screenshot of Skyline software for MRM optimization. Left: the list of selected peptides and fragment ions to create MRM method. Middle: the XICs of one MRM transition with different CE values for MRM optimization. The CE associated with the largest peak area is the optimal CE value. Right: the representative MS/MS spectra from the imported peptide library. The most abundant fragment ions (ranked and color labeled) are listed as the fragment ions in the left panel for MRM method generation.

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Protein quantification

The UPS protein serial dilution samples are analyzed on a

SCIEX QTRAP 6500+ LC-MS/MS System with the optimized

Scheduled MRM Algorithm method. The lower limit of

quantification (LLOQ) and linear dynamic range (LDR) was

investigated. The LLOQ is reported in the format of ppm, and the

amount of NISTmAb per injection is consistent as 20 µg. All 48

proteins are quantified using this 8 min LC-MRM method with

solid confirmation (2 peptides per protein for most proteins, 2

transitions per peptide). In summary, 23 out of 48 proteins (48%)

can be quantified at 0.09-1 ppm and 24 out of 48 proteins (50%)

can be quantified at 1-5 ppm (Figure 5). Accuracies of all

peptides are 85-115% and the CV%s are within 15%.

Representative XICs of UPS proteins at their LLOQ levels,

calibration curves, and quantification results are shown in Figure

6, 7 and 8.

Figure 5. The summary of LLOQ of UPS proteins. 23 out of 48 proteins (48%) have LLOQs between 0.09 and 1 ppm, 24 out of 48 proteins (50%) have LLOQs between 1 and 5 ppm, 1 protein (small protein with limited peptide selection) have LLOQ larger than 5 ppm.

Figure 7. Representative quantification summary for three proteins (insulin-like growth factor II, serum albumin, serotransferrin, [apotransferrin]). Concentration ranges (unit is ppm), CV% and accuracies are listed.

Figure 6. The XICs of the representative HCPs at their LLOQs. UniProt accession numbers and LLOQ concentrations are listed. The data are processed by using the Analytics function in SCIEX OS Software 1.7.

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Conclusions

A targeted HCP quantification workflow using the SCIEX QTRAP

6500+ LC-MS/MS System is presented here, demonstrating high

sensitivity, analysis throughput, robustness and multiplexing

capability.

• High resolution MS/MS peptide library generated by IDA

analysis offers reliable signature peptide selection

• A seamless MRM method development workflow with the

combination of Skyline and SCIEX OS Software allows fast

and robust MRM method optimization

• A total of 48 proteins are quantified in an 8 min LC-MS/MS run

with solid confirmation (4 transitions per protein)

• Superior sensitivity is achieved, with half of target proteins

quantified at 0.09-1 ppm level, the other half quantified at 1-5

ppm level

Figure 8. Representative calibration curves for HCPs. Dynamic ranges covers three orders of magnitude or more for most analytes.

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Table A1. List of UPS proteins (protein names and uniProt accession numbers are included).

uniProt Accession Number

UniProt Protein Name [Synonym]

uniProt Accession Number UniProt Protein Name [Synonym]

P00915 Carbonic anhydrase 1 P55957 BH3 Interacting domain death agonist [BID]

P00918 Carbonic anhydrase 2 O76070 Gamma-synuclein

P01031 Complement C5 [Complement C5a] P08263 Glutathione S-transferase A1 [GST A1-1]

P69905 Hemoglobin alpha chain P01344 Insulin-like growth factor II

P68871 Hemoglobin beta chain P01127 Platelet-derived growth factor B chain

P41159 Leptin P10599 Thioredoxin

P02768 Serum Albumin P99999 Cytochrome c[Apocytochrome c]

P62988 Ubiquitin P06396 Gelsolin

P04040 Catalase P09211 Glutathione S-transferase P [GST]

P00167 Cytochrome b5 P01112 GTPase HRas [Ras protein]

P01133 Epidermal Growth Factor P01579 Interferon gamma (IFN-gamma)

P02144 Myoglobin C P02787 Serotransferrin [Apotransferrin]

P15559 NAD(P)H dehydrogenase [quinone] 1 [DT Diaphorase] C O00762 Ubiquitin-conjugating enzyme E2 C [UbcH10]

P62937 Peptidyl-prolyl cis-trans isomerase A [Cyclophilin A] P51965 Ubiquitin-conjugating enzyme E2 E1 [UbcH6]

Q06830 Peroxiredoxin 1 P08758 Annexin A 5

P63165 Small ubiquitin-related modifier 1 [SUMO-1] P02741 C-reactive protein

P00709 Alpha-lactalbumin P05413 Fatty acid-binding protein

P06732 Creatine kinase M-type [CK-MM] P10145 Interleukin-8

P12081 Histidyl-tRNA synthetase [Jo-1] P02788 Lactotransferrin

P61626 Lysozyme C P10636 Microtubule-associated protein tau [Tau protein]

Q15843 Neddylin [Nedd8] P00441 Superoxide dismutase [Cu-Zn]

P02753 Retinol-binding protein P01375 Tumor necrosis factor [TNF-alpha]

P16083 Ribosyldihydronicotinamide dehydrogenase [quinone] [Quinone oxidoreductase 2] [NQO2]

P63279 Ubiquitin-conjugating enzyme E2 I [UbcH9]

P01008 tithrombin-III

P61769 Beta-2-microglobulin

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